Surfactants are used in a variety of applications. For example, surfactants are used commercially for cleaning manufactured items, removing paints, chemical processing, for use in emulsion polymerization, solubilizing drugs, purifying proteins, and various bioanalytical applications.
In addition, surfactants have been employed in chemical alteration reactions, e.g., reduction or alkylation, involving biomolecules, such as proteins, for solubilization, or the surfactants are present in the reaction as an artifact of the process of preparation, e.g., electrophoresis. Reactions, e.g., reduction and alkylation, of large proteins are important steps for in-solution digestion because of their ability to increase the number of peptide fragments. Organic salts, such as urea and detergents, e.g., sodium dodecylsulfate (SDS), are commonly used to solubilize protein mixtures before reduction and alkylation. However, urea and SDS inhibit trypsin activity, and therefore their concentrations have to be diluted prior to in-solution trypsin digestions. Additionally, it is known that if digestion, in which urea has been utilized as a solubilizing agent, is allowed to proceed for too long, the urea will act upon and modify the protein, making analysis of peptide fragments more difficult and inaccurate.
Furthermore, SDS and urea are also known to suppress the MS signal if they are not removed. The additional sample preparation steps that are required for current methodologies decrease the reliability and sensitivity of the analysis, especially for low abundance proteins.
Another particular bioanalytical application that uses surfactants is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the past three decades, SDS-PAGE has been widely used as a simple and relatively rapid tool for analysis and purification of large molecules such as proteins (U. K. Laemmli, Nature 227, 680-685, 1970). Sodium dodecylsulfate (SDS) is an anionic surfactant that denatures proteins by forming a stable complex. Upon denaturation, SDS binds to most proteins and peptides in a constant weight ratio of about 1.4:1. As a result, the SDS-protein complexes have almost identical charge densities and therefore migrate in a polyacrylamide gel according to molecular weight. If the gel is of the correct porosity, a plot of log Mw vs. relative mobility, Rf, results in a linear relationship. The band intensity after staining is a rough indicator of the amount present in the sample. When coupled with another electrophoretic technique, isoelectricfocusing, SDS-PAGE can separate complex mixtures into several hundred discrete components.
The ability to estimate the size and amount of a protein has led to various applications of SDS-PAGE. However, there are some drawbacks to the technology. For example, it is very difficult to use mass spectrometry to monitor and analyze samples from SDS-PAGE separations because SDS interferes with the sensitivity of mass spectrometry detection. Furthermore, it is very difficult to separate SDS from SDS/protein complex since SDS is a surfactant that forms emulsions.
Protein digestion to produce protein fragments is an important aspect of protein characterization. Currently, the rate-limiting step in mass spectrometric analysis of protein fragments is the extended time required for digestion, e.g., typically 12 hours or more as in the case of trypsin digestion of proteins. Furthermore, the large amounts of trypsin required in current protocols can result in increased background noise due to trypsin autolysis. In addition, the current approaches to trypsin digestion result in mass spectrometric identification of only a limited number of the peptide fragments, e.g., about 60%.
It is also known in the art that trypsin digestion can be accelerated by: (1) performing the digestion at elevated temperatures (Anal. Chem. 2001, 73, 2558-2564); (2) in the presence of certain organic solvents (Anal. Chem. 2001, 73, 2682-2685); or (3) using immobilized trypsin. However, these methods often result in miscleavages, or are difficult to reproduce. Therefore, when it is desirable to generate a reproducible peptide map, the preferred methodology is overnight incubation, often done at low temperature. Nevertheless, a method for enhancing (e.g., acceleration with high reproducibility and low miscleavage) the digestion of proteins is desired.